While the role of mitochondria as the powerhouse of the cell is widely appreciated, mitochondria have other roles in cardiomyocytes (CMs) that are less widely known and understood. These energy-independent roles of mitochondria in both heart development and heart disease are highlighted by an X-linked mitochondrial car- diomyopathy known as Barth Syndrome (BTHS; OMIM #302060). In BTHS, mutation of the acyltransferase Tafazzin (TAZ) depletes the inner mitochondrial membrane (IMM) of its normal complement of cardiolipin (CL), its principle phospholipid.1,2 As a result, the IMM environment does not support normal function of the electron transport chain, the F1F0ATP synthase, and other key mitochondrial protein complexes. Abnormalities of cardi- olipin have been implicated in a range of more common cardiovascular conditions, including ischemia/reperfu- sion and diabetes. Studies of TAZ function and CL metabolism thus have ramifications for common cardiovascular diseases. We studied an induced pluripotent stem cell (iPSC)-derived CM (iPSC-CM) model of BTHS and found that BTHS iPSC-CMs do not mature normally. Sarcomeres did not assembly properly, and myocardial tissues assembled from the diseased iPSC-CMs contracted weakly. These defects were not res- cued by conditions that normalized ATP levels. Rather, BTHS iPSC-CM mitochondria generated excessive ROS, and ROS normalization rescued BTHS iPSC-CM structural and functional defects. This proposal builds on these findings, using Barth syndrome as a window into novel aspects of mitochondrial regulation of cardiac development and function. At the same time, we will work towards therapy for this disease. We propose the fol- lowing Specific Aims: Aim 1. To define mechanisms through which mitochondrial abnormalities in BTHS impair CM function, focusing on ROS as a mechanism by which mitochondrial disease perturbs cardiomyocyte calci- um handling. Aim 2. To evaluate mitochondrial regulation of cardiac morphogenesis and cardiomyocyte matu- ration. We will study normal LV compaction and cardiomyocyte maturation and how they are disrupted by mitochondrial dysfunction. Aim 3. To perform preclinical proof-of-concept studies of targeted therapies for Barth syndrome. This study will provide fundamental information on cardiolipin and mitochondrial function in heart development and heart disease. The studies will have direct relevance to the pathogenesis of BTHS as well as more common cardiovascular diseases and will inform development of targeted therapy for BTHS.